This disclosure relates to an improved method of identifying proteins of interest, e.g., binding molecules such as antibodies or fragments thereof, in eukaryotic cells, and in particular, improved method of producing protein libraries, e.g., antibody heavy and/or light chain libraries for expression in eukaryotic cells.
Eukaryotic Expression Libraries
A basic tool in the field of molecular biology is the conversion of poly (A)+ mRNA to double-stranded (ds) cDNA, which then can be inserted into a cloning vector and expressed in an appropriate host cell. A method common to many cDNA cloning strategies involves the construction of a “cDNA library” which is a collection of cDNA clones derived from the poly(A)+ mRNA derived from a cell of the organism of interest. For example, in order to isolate cDNAs which express immunoglobulin or antibody subunit polypeptides, a cDNA library might be prepared from pre-B cells, B cells, or plasma cells. Methods of constructing cDNA libraries in different expression vectors, including filamentous bacteriophage, bacteriophage lambda, cosmids, and plasmid vectors, are known.
Many different methods of isolating target genes from cDNA libraries have been utilized, with varying success. These include, for example, the use of nucleic acid hybridization probes, which are labeled nucleic acid fragments having sequences complementary to the DNA sequence of the target gene. When this method is applied to cDNA clones in transformed bacterial hosts, colonies or plaques hybridizing strongly to the probe are likely to contain the target DNA sequences. Hybridization methods, however, do not require, and do not measure, whether a particular cDNA clone is expressed. Alternative screening methods rely on expression in the bacterial host, for example, colonies or plaques can be screened by immunoassay for binding to antibodies raised against the protein of interest. Assays for expression in bacterial hosts are often impeded, however, because, e.g., the protein is not be sufficiently expressed in bacterial hosts, the protein is expressed in the wrong conformation, or the protein is not processed, and/or transported as it would in a eukaryotic system. Many of these problems have been encountered in attempts to produce antibody molecules in bacterial hosts, as alluded to above.
Accordingly, use of eukaryotic, e.g., yeast or mammalian expression libraries to isolate cDNAs encoding proteins of interest, e.g., binding molecules such as antibodies or antigen-binding fragments thereof, offer several advantages over bacterial libraries. For example, binding molecules such as antibodies or antigen-binding fragments thereof, and subunits thereof, expressed in eukaryotic hosts can be functional and can undergo typical eukaryotic posttranslational modification. A protein ordinarily transported through the intracellular membrane system to the cell surface can complete the transport process. Further, use of a eukaryotic system makes it possible to isolate polynucleotides encoding proteins of interest based on functional expression of eukaryotic RNA or protein. For example, binding molecules such as antibodies or antigen-binding fragments thereof can be isolated based on their specificity for a given antigen. See, e.g., U.S. Pat. No. 7,858,559, U.S. Patent Appl. Publ. No. 2016-0152971, and U.S. Prov. Appl. No. 62/326,501, filed on Apr. 22, 2016, each of which is incorporated herein by reference in its entirety. See also Smith et al., Nature Medicine 7:967-972 (2001).
Poxvirus Vectors
Poxvirus vectors are used extensively as expression vehicles for protein and antigen expression in eukaryotic cells. The ease of cloning and propagating vaccinia in a variety of host cells has led to the widespread use of poxvirus vectors for expression of foreign protein and as vaccine delivery vehicles (Moss, B., Science 252:1662-7 (1991)).
Traditionally, poxvirus vectors were not used to identify unknown genes of interest from a complex population of clones, because a high efficiency, high titer-producing method of cloning did not exist for pox viruses. The present inventors, however, developed a method for generating diverse cDNA libraries in recombinant poxviruses using tri-molecular recombination. See, e.g., Zauderer, U.S. Pat. No. 6,706,477, issued Mar. 16, 2004, and Zauderer et al., U.S. Pat. No. 7,858,559, issued Dec. 28, 2010, each of which is incorporated herein by reference in its entirety.
Tri-molecular recombination by itself is a high efficiency, high titer-producing method for producing recombinant poxviruses. There remains a need to further enhance the process of using tri-molecular recombination to yield an even greater amount of recombinant viruses.